The occurrence of Shiga toxin-producing Escherichia coli (STEC) was studied on four cattle farms. STEC were detected in 29-82% of the cattle. STEC with additional EHEC markers were detected on all farms. The occurrence of the complete virulence marker pattern (stx1 and/or stx2, eae, EHEC(hlyA), katP, espP) was correlated with the presence of known STEC serotypes. STEC O26:H11 and O165:H25 with the complete pattern of virulence markers were the most prevalent. STEC O157 (H7/H-) STEC O103:H2 and STEC O145:H- were found sporadically. Five clonal subgroups of the STEC O26:H11 isolates were identified by pulsed-field gel electrophoresis. STEC O26:H11 were present in three groups of cattle. This serotype was detected in a single group over the entire fattening period. Most STEC O26:H11 with the complete pattern of potential virulence markers were found in clinically healthy cattle. These animals may represent a risk factor for farmers and consumers.

Antigen capture enzyme-linked immunosorbent assays (ELISAs) for the detection of Stx1 and/or Stx2 in cattle feces were validated in comparison to the Vero cell cytotoxicity neutralization test (as a "gold standard") applied in the course of a monitoring program for Shiga toxin-producing Escherichia coli in German cattle herds as a prescreening test and compared to MK1/MK2 PCR as an alternative prescreening test.Several enzyme-linked immunosorbent assays (ELISAs) have been developed for the detection of Shiga toxin-producing Escherichia coli (STEC), some of which use the P1 glycoprotein from hydatid cyst fluid, or Gb3, for Shiga toxin capture (1, 2, 16). Recently, monoclonal antibodies (MAbs) to verocytotoxins Stx1 and Stx2 were used to examine animal feces (3). Mitomycin C can enhance Stx production in E. coli and was applied to increase the ELISA sensitivity (12). We describe the validation of ELISAs for the detection of Stx1 and/or Stx2 in cattle feces. These ELISAs were used in a monitoring program as a prescreening test and compared to MK1/MK2 PCR (13).A total of 100 E. coli field strains isolated from cattle were tested in the STEC-ELISAs, the Vero cell cytotoxicity assay (VCA), and the Vero cell cytotoxicity neutralization assay (VCNA) which served as the "gold standard." The assays were performed as described by Konowalchuk et al. (14), with some modifications (8, 17). The cytopathic effect (CPE) and the neutralization of the CPE were measured by determining cell densities in a Coulter Z2 counter (Coulter Electronics Inc., Miami, Fla.). The specificity of the VCA for the toxin production of E. coli field isolates was determined by neutralization of the CPE on Vero cells (ATCC CRL1587) by MAbs 13C4 (ATCC CRL1794) and 11E10 (ATCC CRL1907).The ELISAs utilized hydatid cyst fluid of Echinococcus granulosus for detecting the Shiga toxins, as described previously (18). A total of 200 l of supernatant of the E. coli field strains or preenriched fecal samples of cattle were directly investigated in this assay. Each sample was tested in quadruplicate. Positive controls (E. coli C600-J1 for Stx1 and E. coli C600-W34 for Stx2) and a negative control (E. coli C600) were tested sixfold on each plate (blanks in each corner). The MAbs 13C4 and 11E10 were used for the specific detection of Stx1 and Stx2. Bound MAbs were detected with goat anti-mouse peroxidase conjugate and 3,3Ј,5,5Јtetramethylbenzidine (TMB; KPL, Gaithersburg, Md.) as the substrate. The reactions were stopped by adding 0.5 M sulfuric acid. Optical densities (ODs) were read at 450 nm with a reference filter at 620 nm. The mean ODs of positive and negative controls and of the samples were calculated from the blank-corrected OD readings (OD value Ϫ blank value), and an index was determined for each sample according to the following equation:index ϭ mean of OD sample Ϫ mean of OD negative control mean of OD positive control Ϫ mean of OD negative controlThe study showed that Stx1-and Stx2-specific ELISAs can be utilized for the detection of these Shiga tox...